Plant Soil (2018) 422:183–194 DOI 10.1007/s11104-017-3241-x REGULAR ARTICLE Fungal endophytes from seeds of invasive, non-native Phragmites australis and their potential role in germination and seedling growth Zackery R. C. Shearin & Matthew Filipek & Rushvi Desai & Wesley A. Bickford & Kurt P. Kowalski & Keith Clay Received: 29 November 2016 /Accepted: 28 March 2017 /Published online: 1 April 2017 # Springer International Publishing Switzerland 2017 Abstract respectively were most prevalent. There was a signifi- Background and aims We characterized fungal endo- cant effect of isolating an endophyte from the seed on phytes of seeds of invasive, non-native Phragmites from seedling growth. three sites in the Great Lakes region to determine if fungal Conclusions These results suggest that many endophyte symbiosis could contribute to invasiveness through their taxa are transmitted in seeds and can increase seed germi- effectsonseedgerminationandseedlinggrowth. nation and seedling growth of invasive Phragmites.The Methods Field-collected seeds were surface sterilized and role of fungal endophytes in host establishment, growth plated on agar to culture endophytes for ITS sequencing. and invasiveness in nature requires further research. Prevalence of specific endophytes from germinated and non-germinated seeds, and from seedlings, was compared. Keywords Phragmites australis . Seed endophytes . Results One-third of 740 seeds yielded endophyte iso- Biological invasion . Germination . Seedling growth . lates. Fifteen taxa were identified with Alternaria sp. Fungi representing 54% of all isolates followed by Phoma sp. (21%) and Penicillium corylophilum (12%). Overall germination of seeds producing an isolate (36%) was significantly higher than seeds not producing an isolate Introduction (20%). Penicillium in particular was strongly associated with increased germination of seeds from one site. Endophytic fungi occur in all plant species thus far ’ Sixty-three isolates and 11 taxa were also obtained from examined (U Ren et al. 2012) where they engage in a 30 seedlings where Phoma, Penicillium and Alternaria wide variety of interactions with their plants hosts. By definition, endophytes are organisms that live inside of plants and do not cause disease symptoms on their host Responsible Editor: Eric B. Nelson. (Wilson 1995). Endophytes can include mutualists that Electronic supplementary material The online version of this enhance host plant fitness, latent pathogens that may or article (doi:10.1007/s11104-017-3241-x) contains supplementary may not express disease symptoms at some point in the material, which is available to authorized users. future, or saprophytes that are inactive until tissue se- Z. R. C. Shearin : M. Filipek : R. Desai : K. Clay (*) nescence (Müller et al. 2001;Osono2006;Kleczewski Department of Biology, Indiana University, Bloomington, IN et al. 2012). Fungal endophytes also exhibit variation in 47405, USA their mechanisms of transmission from one host to e-mail: [email protected] another including strict vertical transmission from ma- W. A. Bickford : K. P. Kowalski ternal plants to seeds (Rodriguez et al. 2009), contagious U.S. Geological Survey, Great Lakes Science Center, 1451 Green transmission from one host plant to another (Clay and Road, Ann Arbor, MI 48105-2807, USA Schardl 2002; Porras-Alfaro and Bayman 2011), or 184 Plant Soil (2018) 422:183–194 infection by spores from environmental sources like On the other hand, fungal pathogens may limit the wind, rain, soil, and leaf litter (Christian et al. 2015). establishment and growth of non-native plant popula- Vertically-transmitted endophytes are predicted to tions (Biotic Resistance Hypothesis) so that they never have a mutualistic association with their host because become established or reach densities high enough to detrimental endophytes should be rapidly purged from displace native species (Mack 1996;Kneveletal.2004; the host population (Ewald 1987; Lipsitch et al. 1995). Parker and Gilbert 2004). However, outside of agricul- For example, many Epichloë endophytes infecting cool- tural species, we have little knowledge of failed inva- season grasses such as tall fescue (Lolium arundinaceum) sions due to pathogenic fungi (Scheffer 1997). are only vertically transmitted through seeds and are Understanding the diversity and impacts of fungi asso- typically mutualistic (Schardl and Clay 1997; ciated with seeds of invasive plant species will help Panaccione et al. 2014). Other endophytes infecting dif- evaluate risks and design effective management ferent groups of plants can also be vertically transmitted strategies. and are potentially mutualistic (Ernst et al. 2003; Phragmites australis (Cav.) Trin. ex Steud.) (hereaf- Hodgson et al. 2014), but they have not been subject to ter Phragmites) is a clonal wetland grass that is distrib- as much past research as Epichloë endophytes. By con- uted worldwide in temperate and subtropical regions trast, many fungi are seed borne (Baker and Smith 1966; (Haslam 1972; Kirk et al. 2011; Guo et al. 2013). Rheeder et al. 1990;Martin1996;Donaldetal.2005) Haplotype M is an aggressive invader that is widespread following contagious infection of developing flowers or in the United States (Saltonstall 2002, 2003). Invasive, seeds by spores. For example, plants arising from seeds non-native Phragmites (hereafter invasive Phragmites) infected by loose smut of barley and wheat (caused by can result in the displacement of native plant and animal Ustilago spp.) produce diseased inflorescences contain- species (Keller 2000; Holdredge and Bertness 2011; ing spore masses that can then contagiously infect flowers Kessler et al. 2011; Price et al. 2013), and alter wetland and seeds of non-diseased plants (Wunderle et al. 2012). hydrology and soils (Windham and Lathrop 1999). With strict vertical transmission, every seed produced by Several mechanisms have been proposed to explain an individual plant could be infected by a single endo- the success of invasive Phragmites, including its poten- phyte whereas in the case of seed-borne fungi each seed tial association with mutualistic fungal endophytes produced by one plant could be independently infected (Kowalski et al. 2015;Clayetal.2016). In a recent by distinct fungi from different sources. study, fungal endophytes associated with leaves, stems, The microbiota of seeds may be an important source and rhizomes of invasive Phragmites were isolated and for colonization of the next generation of seedlings and sequenced to reveal significant differences in endophyte adult plants (Hodgson et al. 2014;Klaedtkeetal.2015; community structure among tissue types and popula- Truyens et al. 2015). The seed microbiota may be espe- tions (Clay et al. 2016). The functional role of these cially important for invasive plant species given that endophyte associations is not known, but one common- there are no pre-existing conspecific populations or ly isolated fungal genus in the Great Lakes region associated microbes present upon initial colonization (Stagonospora, Clay et al. 2016) has been reported to of a new habitat (Newcombe et al. 2009). Vertically- be vertically transmitted through seeds and to enhance transmitted, mutualistic fungi may enhance the ability of Phragmites growth in Europe (Ernst et al. 2003). invasive host plants to colonize new habitats, compete The goals of this study were to isolate and identify with established species, and resist parasites and patho- fungal endophytes from seeds and seedlings of invasive gens (Aschehoug et al. 2012; Saikkonen et al. 2013). Phragmites from the Great Lakes region of the United For example, infection by vertically-transmitted endo- States and to explore their potential impacts on seed phytic fungi was found to be responsible for the com- germination and seedling growth. While our results are petitive advantage of the invasive tall fescue grass derived from a limited number of sites and an unknown (Rudgers et al. 2005; Rudgers and Clay 2008). By number of established Phragmites genotypes, they rep- contrast, the absence of mutualistic fungi can limit the resent the first critical analysis of fungal endophytes in success of non-native species. For example, plantings of the seeds of this important invasive species. Further, pines (Pinus sp.) in the southern hemisphere were ini- because of previous results based on endophyte isola- tially unsuccessful until compatible ectomycorrhizal tions from leaf, stem, and rhizome tissues of adult symbionts were also introduced (Pringle et al. 2009). Phragmites, a general comparison of endophyte Plant Soil (2018) 422:183–194 185 diversity in seeds and seedlings with adult plants could site. It is likely that different patches represented differ- potentially provide insights into the ability of endo- ent genotypes (or clones), but we did not do any diag- phytes to be transmitted in seeds and colonize ensuing nostic tests to identify distinct Phragmites genotypes. plants. In particular, we addressed the following Seed-borne endophytes or those from environmental questions: sources could be unrelated to plant genotype whereas seed-transmitted endophytes could be more specific to Do fungal endophytes colonize Phragmites seeds individual genotypes. Upon return to the lab, a subset of and if so, what fungi? florets were dissected to verify viable seed was present. Do fungal endophytes in seeds colonize Inflorescences were then wrapped in filter paper, placed Phragmites seedlings and if so, what fungi? into trays, and buried